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A MAPK cascade in Arabidopsis innate immunity

New England Arabidopsis Meeting - April 3rd 2002. A MAPK cascade in Arabidopsis innate immunity. Guillaume Tena. Signal. Input. The prototypical MAPK cascade. Sensor. MAPKKK. Filter Amplify. MAPKK. MAPK. Primary Response Genes. Output. Secondary Response Genes.

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A MAPK cascade in Arabidopsis innate immunity

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  1. New England Arabidopsis Meeting - April 3rd 2002 A MAPK cascade in Arabidopsis innate immunity Guillaume Tena

  2. Signal Input The prototypical MAPK cascade Sensor MAPKKK Filter Amplify MAPKK MAPK Primary Response Genes Output Secondary Response Genes

  3. How many genes involved?

  4. 20 MAPKs in Arabidopsis (structurally homogenous) B MPK12 A MPK5 MPK4 MPK6 MPK3 MPK11 MPK10 MPK13 MPK1 MPK15 C MPK2 MPK8 MPK7 MPK9 MPK14 MPK20 MPK17 D MPK16 MPK18 MPK19

  5. 10 MAPKKs in Arabidopsis (structurally homogenous) MKK8 MKK7 MKK9 MKK5 MKK4 MKK10 MKK2 MKK3 MKK1 MKK6 D C A B

  6. A 60 MAPKKK in Arabidopsis MAP3Ke1 MAP3Ke2 MEKK4 MEKK3 MEKK2 At1g63700 MAP3Kg MAP3Ka MEKK1 ANP2 ANP1 At5g01850 At5g50180 ATN1 At5g40540 ANP3 At5g66710 At3g50720 C1 At4g18950 ATN1-like At4g38470 At3g58760 At3g59830 At2g17700 At4g35780 At2g43850 At2g24360 At4g31170 At2g31800 At3g46920 At1g14000 At3g06630 At5g57610 At3g06620 At3g06640 At2g35050 MAP3Ke3 At3g24720 At1g04700 At1g67890 At1g16270 MAP3Kd4 CTR1 At1g79570 MRK1 B C MAP3Kd5 At3g01490 At5g50000 At3g22750 At4g14780 At3g58640 MAP3Kt2 At1g62400 MAP3Kt1 At1g73660 At1g18160 At3g46930 MAP3Kd1 EDR1 At5g58950

  7. Structural heterogeneity of plant MAPKKKs

  8. Complexity of MAPK networks 20 x 10 x 60 = a lot of virtual combinations How to work with such a high number of possibilities? a simplified biological system possibility to do hundreds of functional analyses quickly

  9. SIMPLIFIED BIOLOGICAL SYSTEM (to quickly select interesting genes for later analyses in whole plant) From an entire plant… to a tissue… to a single type of cell Arabidopsis mesophyll cells protoplasts: an homogenous and easily manipulable material Complexity

  10. Functional Genomic Analyses of Plant Signal Transduction in Arabidopsis protoplasts

  11. The early defense responses

  12. 1. Local defense First line of protection = innate immunity Very early detection of invaders in infected cells through specific or generic interactions Restricts pathogen growth and spread (PCD, production of defensive molecules) Pathogen 2. Systemic defense Protects non-infected tissues from secondary infection

  13. SIMPLIFIED PATHOGENIC SIGNAL From bacterium… to flagellum… to flagellin… to flag22 Biologically active synthetic peptide acting as a general elicitor: just one pathway involved QRLSTGSRINSAKDDAAGLQIA Complexity

  14. The starting point model (From previous work in T. Boller’s lab)

  15. A Model for Defense Gene Induction (From previous work in T. Boller’s lab) Signal Transduction Inactive TF Active TF TF FLS2 Flag22 (LRR) (Kinase) (MAPK6) Early Defense Genes Late Defense Genes

  16. Are early defense responses activated by flag22 in our protoplast system? Flg22 receptor MAPKKK MAPKK MPK Genes

  17. …MAPKsin 10 minutes (In-gel kinase assay, MBP as substrate) Flg22 receptor MAPKKK MAPKK MPK Flg22 activates endogenous… Genes … Genes in 30 minutes (RT-PCR on genes identified from a substracted cDNA library) Flg22 receptor MAPKKK MAPKK MPK Genes

  18. Which MAP kinases?

  19. ... MAPK 3 and 6 (Immunocomplex assay, MBP as substrate) (35S Methionine labelling) Flg22 receptor MAPKKK MAPKK MPK3/6 Genes Flg22 activates transfected… … Promoters+LUC Flg22 receptor MAPKKK MAPKK MPK Genes

  20. Through which receptor?

  21. ... MAPK3/6 activation by flg22 (protoplasts from fls2 mutant and wild-type) (Immunocomplex assay, MBP as substrate) (35S Methionine labelling) Flg22 FLS2 MAPKKK MAPKK MPK3/6 Genes FLS2 receptor is needed for… ... Gene activation by flg22 Flg22 FLS2 MAPKKK MAPKK MPK Genes

  22. What happens if we block the MAPK? (there is no specific inhibitor of plant MAPKs : use a mouse MAPK phosphatase)

  23. ... totally the MAPK3/6 activation by flg22 (protoplasts from wild-type) (Immunocomplex assay, MBP as substrate) (35S Methionine labelling) Flg22 FLS2 MAPKKK MAPKK MKP1 MPK3/6 Genes A MAPK phosphatase blocks… … half of the gene activation by flg22 Flg22 FLS2 MAPKKK ? MAPKK MKP1 MPK3/6 Genes GST1

  24. Probably two pathways involved in the response: one MAPK dependent (blocked by MKP1, MKK inhibitors, dominant-negative) one MAPK independent (unknown)

  25. After genes, signal, receptor, MAPK Next step: MAPKK

  26. Manipulation of MAPKK activity Kinase-dead mutant and constitutively active (dominant-negative) M D or E

  27. Constitutively active MKK5 activates MAPK 3 and 6 Without the need for Flg22 signal Flg22 FLS2 MAPKKK MKK5 MPK3/6 Genes (Immunocomplex assay, MBP as substrate) (35S Methionine labelling)

  28. ... The MAPK3/6 activation (protoplasts from wild-type) (Immunocomplex assay, MBP as substrate) (35S Methionine labelling) Flg22 FLS2 MAPKKK MKK4/5 MPK3/6 Genes MKK4 and 5 are redundant for... … Gene activation … Downstream of the receptor Flg22 FLS2 MAPKKK MKK4/5 MPK3/6 Genes

  29. Next step: MAPKKK

  30. Kinase Manipulation of MAPKKK activity Substrate Removal of regulatory domains (auto-inhibitory, interaction with upstream factors, …) constitutively active MAPKKK Kinase Substrate

  31. ... MKK5 (Immunocomplex assay, GST-MPK6km as substrate) (35S Methionine labelling) ... MPK3/6 (Immunocomplex assay, MBP as substrate) Flg22 FLS2 MEKK1 MAPK MKK4/5 MEKK1 MPK3/6 Genes MEKK1 activates... … Downstream gene activation No need for the receptor Flg22 FLS2 MEKK1 MKK4/5 MPK3/6 Genes

  32. Going deeper in the pathway: The effect of WRKY29 itself

  33. N C Target Sequence : W box (T)(T)TGAC(C/T) General Features of WRKY Transcription Factors WRKY NLS domain ~ 60 a.a. ---- WRKY ------------ C ----- C ----------------- H - H -- Zinc-finger motif (Trp-Arg-Lys-Tyr)

  34. WRKY29::GFP has a nuclear localization

  35. WRKY29 activates its own promoter and is a member of a sub-family containing WRKY22 as well … and this happens downstream of FLS2

  36. Final signaling pathway analyzed in protoplasts Signal Flg22 Receptor FLS2 MEKK1 MAPK cascade MKK4/5 MPK3/6 WRKY29/22 Early responses early genes (FRK1) late genes (PR)

  37. What about in plants?

  38. Complexity of interactions Going back to the real world Flag22 QRLSTGSRINSAKDDAAGLQIA

  39. … bacterial infection with Pseudomonas syringae 100 times reduction of bacterial growth after 4 days Agrobacterium-mediated transient transformation of Arabidopsis leaves to activate the pathway protects from... … fungal infection with Botrytis cinerea MKK1a (control) MKK4a

  40. Fungal resistance of transgenic Arabidopsis MEKK1 MKK4a WRKY29 vector

  41. Conclusion: the innate immune system is very conserved between Arabidopsis, mammals and Drosophila

  42. Conservation of innate immunity signaling pathways

  43. Acknowledgments Jen Sheen’s gang Fred Ausubel’s gang Everybody Joulia Plotnikova Tsuneaki Asai Thomas Boller Lourdes Gomez-Gomez For the FLS2 seeds Kazuya Ichimura Kazuo Shinozaki For the MAPKKK slide

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